Although much work in recent years has been focused on the role of lumenal ER chaperones in the folding and assembly of secretory proteins, many of these proteins also possess rather long cytosolic portions. It is assumed that they also require the action of chaperones, but this has not been investigated in any detail. Members of the ABC protein family have very long cytosolic domains that encompass a major portion of the protein. It is well recognized that dnaJ homologues interact with dnaK homologues (hsp70s) to promote the folding of substrate proteins. Cytosolic, ER localized, dnaJ homologues (hsp40s) have been identified in yeast and mammals. It is the hypothesis of this proposal that YDJ-1 (yeast) and HDJ- 1 and -2 (mammalian) collaborate with cytosolic hsp70 to fold the cytosolic tail of ER membrane proteins. In the first aim, the P.I. will use genetic and biochemical techniques to examine the role of YDJ-1 and hsp70 and Ste6 folding in S. cerevisiae. In the second aim, he will develop a cell free system to characterize reaction intermediates in the pathway for CFTR folding. In the third aim, purified components will be employed to determine the molecular mechanism by which hsp40/hsp70 chaperone pairs facilitate the folding of soluble cytosolic domains on membrane proteins.